实验概要

Extract the total RNA from animal cells by using RNeasy Mini Kit (QIAGEN No.74104)

主要试剂

SDS based extraction solution

实验步骤

1.  Harvest cells.

1)   Trypsinize and collect cells, aspirate medium, and wash cells with PBS twice.

2)   Determine the number of cells.

3)    Transfer cells to an RNase-free centrifuge tube and pellet by  centrifugation at    300 x g for 5 min. (In our lab, use 1800rpm in the  eppendorf centrifuge 5415D). Cell pellets can be stored at –70°C for  later use or used directly in the procedure.

2.   Lyse cells

 1) Thaw frozen cell pellets slightly.

 2)  Add Buffer RLT (Add 10 µl –ME per 1 ml Buffer RLT). Usually, If number  of pelleted cells <5 x 106, add 350µl Buffer RLT.If number of  pelleted cells is  around 5 x 106 – 1 x 107, add 600µl Buffer RLT.

 3) Vortex or pipet to mix.

3.   Homogenize the sample.

       It is best to use a rotor–stator homogenizer to homogenize cells for 30  s. If not, pass the lysate at least 5 times through a needle fitted to  an RNase-free syringe.

4.   Add 1 volume (usually 350 µl or 600 µl) 70% ethanol, mix using pipet. Do not centrifuge.

5.    Take up to 700µl of that solution and run through a supplied mini  column for 15 seconds at 12,000 rpm. Repeat the previous step until all  of the solution has run through the mini column.

6.   Add 350 µl Buffer RW1 into the column, and centrifuge for 15 s at 12,000rpm to wash. Discard the flow-through.

7.    Add 10 µl DNase I stock solution (Dissolve the solid DNase I (1500  Kunitz units) in 550 µl of the RNase-free water.) to 70 µl Buffer RDD.  Mix gently.

8.   Add the DNase I incubation mix (80 µl) directly onto the RNeasy membrane, 20–30°C for 15 min.

9.   Run 700µl RW1 through the mini column, 15 seconds at 12,000 rpm.

10.   Change out the collection tube under the mini column. Run 500µl RPE  (ethanol added) through the column, 15 seconds at 12,000 rpm.

11.  Spin another 500µl RPE through the column for 2 minutes at maximum speed (13,200 rpm for us).

12.  Discard the flow-through. Spin another 1 minute at maximum speed (13,200 rpm for us) to ensure that the column is dry.

13.  Add 30-50µl RNase-free H₂O directly to the filter of the column, and spin for 1 minute at 12,000 rpm.

注意事项

1.        All steps performed at room temperature. 

2.        RNA can be stored at –80°C for short time.

3.        At step 8, it is needed to prolong the treated time for cell >5 x 106.